Safe, effective, and reversible methods for contraception are necessary to address the 85 million unplanned pregnancies that occur worldwide each year. In addition to the negative impact of these unintended pregnancies on global sustainability, nearly one-fifth of these cases are terminated through unsafe abortions with significant risks to women?s health. Since the invention of ?the Pill? in the 1950s, the vast majority of birth control options have been female-directed, including estrogen or progestin treatments, barrier methods, intrauterine devices, and tubal ligation. In contrast, men remain limited to condoms and vasectomy, which have high failure rates and incomplete reversibility, respectively. Pharmacological strategies for male contraception would help achieve parity with current female-directed options. However, hormone-based therapies can lead to metabolic disorders, mood changes, thrombosis, acne, and testicular degeneration. Non-hormonal agents in development such as retinoic acid signaling antagonists, lonidamine derivatives, and bromodomain testis-specific protein inhibitors can also have undesirable on-target side effects. Identifying new regulators of sperm development and function will be necessary to bridge this gap, and druggable testis-specific proteins are especially attractive targets. Our project focuses on one signaling protein that exemplifies this paradigm: HIPK4, a member of the homeodomain-interacting protein kinase family that is expressed in developing sperm. We observe that male mice lacking HIPK4 function are infertile but otherwise appear to have normal development, physiology, and behavior. HIPK4-deficient mice exhibit spermatogenic defects that are consistent with oligoasthenoteratozoospermia, and their misshapen sperm are incompetent for in vitro fertilization. Our investigations further indicate that HIPK4 regulates actin-driven head shaping during spermatid elongation. Our findings underscore the potential of small-molecule HIPK4 inhibitors as non-hormonal male contraceptives, particularly antagonists that target regions outside of the conserved ATP-binding pocket. Toward this goal, we will develop allosteric HIPK4 inhibitors and evaluate their effects on spermiogenesis and male fertility in animal models. The R61 phase of this project will focus on establishing a workflow for identifying and characterizing allosteric HIPK4 antagonists, including a primary protein thermal shift (PTS) assay (R61 Aim 1) and secondary/tertiary assays of inhibitor potency and selectivity (R61 Aim 2). We will also develop in silico and crystallographic protocols for studying the structural basis of HIPK4 inhibition (R61 Aim 3). After completing these milestones, we will pursue the R33 phase of this project, which includes a large-scale, high-throughput PTS screen for allosteric HIPK4 inhibitors (R33 Aim 1) and hit-to-lead optimization through medicinal chemistry and structure-based design (R33 Aim 2). We will then evaluate HIPK4 antagonists in animal models to determine their safety, efficacy, and reversibility as male contraceptives (R33 Aim 3).